Twospotted spider mite, Tetranychus urticae (Koch), has become a significant early season pest of cotton, Gossypium hirsutum L., in the midsouthern U.S. Sixteen experiments were conducted across the midsouthern U. S. to determine the impact of twospotted spider mite infestation timing on cotton injury, stunting, and yields. Twospotted spider mites from a greenhouse colony were used to initiate infestations at the three-leaf stage, at first flower, and at 200 heat unit intervals after first flower. Twospotted spider mite injury on a scale of zero to five (0 = no injury, 5 = severe injury), plant stunting, and final cotton yields were measured. In general, all infestation timings had higher injury ratings compared to the uninfested control. The highest injury ratings were observed for the three-leaf and first flower infestations. Additionally, infestations at the three-leaf stage caused more plant stunting than later infestation timings. In most of the experiments, the three-leaf infestation resulted in significant stunting of cotton plants. For cotton yields, early infestations caused the greatest yield losses. Significant yield losses compared to the untreated control were observed for infestations initiated up to first flower plus 800 heat units. These results suggest that cotton should be protected from twospotted spider mite infestations beyond that point in the growing season. Results from this experiment will be used to improve integrated pest management of twospotted spider mite in cotton.